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Ethanol induced modulation of microglial P2X7 receptor expression and its role in neuroinflammation

ETHANOL INDUCED MODULATION OF MICROGLIAL P2X7 RECEPTOR EXPRESSION AND ITS ROLE IN NEUROINFLAMMATION
by
Sushmitha Gururaj
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2012
Copyright 2012 Sushmitha Gururaj

The present work builds upon a central hypothesis that ATP-gated purinergic P2X7 receptors (P2X7Rs) play an important role in causing ethanol-induced neuroinflammation and neurodegeneration. A distinctive set of properties set the P2X7R apart from other members of the P2X superfamily, including activation by pathologically significant levels of ATP and structural uniqueness that allows participation in a multitude of signal transduction pathways. Further, P2X7Rs are mainly localized to microglia in the brain and building evidence suggests that they play a critical role in the activation of these cells. Recently published studies have suggested that P2X7R signaling in microglia contributes to neurodegenerative pathologies through a neuroinflammatory response involving the production of pro-inflammatory cytokines such as Interleukin -1β (IL-1β). Despite the fact that alcohol abuse induces microglial activation and neuroinflammation similar to neurodegenerative pathologies, the role of the P2X7R in alcohol-induced neuroinflammation and brain damage has not been investigated so far. ❧ Three Specific Aims were set forth as initial steps to test the central hypothesis with the objective of taking us closer to understanding the mechanism by which alcohol-induced changes in P2X7R expression and function can cause or modulate the resulting inflammatory cascade, which may further cause or modulate the resultant alcohol-induced brain damage. Aim 1 tested the effects of ethanol exposure on P2X7R expression in different brain regions of C57BL mice using two different drinking paradigms: continuous access two-bottle choice and continuous access single bottle no choice. Using Western immunoblotting, these studies demonstrated that ethanol exposure had a differential effect on P2X7R expression in brain regions depending on the amount and duration of ethanol exposure. Low level of ethanol exposure (10 E) for a short period (10 d) caused an up-regulation of P2X7R in alcohol sensitive brain regions, whereas exposure to higher ethanol levels (20 E) for a longer period of time (3months) reversed the effect to down-regulation of P2X7R. Aim 2 tested effects of ethanol on P2X7R expression levels in the BV2 murine microglial cell line (in vitro) using Western immunoblotting. These studies found that ethanol up-regulated microglial P2X7R expression in both the acute (24 h) and chronic (7 d) models. Aim 3 studies tested the effects of ethanol on the P2X7R-mediated release of IL-1β in BV2 microglial cells in the absence and presence of an inflammatory stimulus, LPS. Using ELISA measurements, the studies demonstrated that ethanol causes an up-regulation in LPS-induced IL-1β production and release upon ATP-activation of P2X7Rs. ❧ Collectively, our findings from the three aims supported our central hypothesis and suggested that ethanol-induced modulation of microglial P2X7R expression mediates a neuroinflammatory response involving IL-1β release, the latter being a known mediator of neurodegeneration. The confirmation of our hypothesis prompted efforts toward an effective way to prevent ethanol-induced neuroinflammatory effects. Thus, Aim 3 also included preliminary studies to inhibit P2X7R expression in BV2 microglia using a lentiviral-mediated shRNA transfer strategy. We were able to achieve successful knockdown with one of the two shRNAs that we generated, and attempts to use this shRNA construct to establish a stable BV2 cell line with long-term P2X7 knockdown are currently being made. This could be used in future studies as a tool to investigate the downstream effects of P2X7R inhibition and identify the signaling mechanisms by which ethanol delivers its toxic neurological effects.

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ETHANOL INDUCED MODULATION OF MICROGLIAL P2X7 RECEPTOR EXPRESSION AND ITS ROLE IN NEUROINFLAMMATION
by
Sushmitha Gururaj
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2012
Copyright 2012 Sushmitha Gururaj